It is the objective of this proposal to determine the X-ray crystal structure at 2.7 Angstroms resolution of inorganic pyrophosphatase from Baker's yeast (PPase). This enzyme which is essential for life, controls the level of pyrophosphate ion. This ion may, in turn, have a central role in controlling the calcification of bones and teeth through its control of the rate of formation and growth of calcium phosphate crystals. The structure of PPase may lead to a general model for phosphate-transfer enzymes which are among the most wide-spread and important enzymes utilized in biological processes. PPase crystallizes in the space group P2, with one dimeric molecule of molecular weight 64,000 in the asymmetric unmit of its unit cell. The X-ray diffraction data of native PPase crystals and two of tis isomorphous heavy atom derivatives have been recorded by rotation photography at a temperature of -50 degrees to their practical resolution limit of 2.7 Angstroms. These data will be scaled together and the phases determined using multiple isomorphous replacement data combined with analmalous dispersion information. The phase information may be further improved by utilizing the redundant information inherent in the noncrystallographic symmetry of the PPase dimer. The structural model of PPase will be determined from the resulting electron density map together with the known amino acid sequence of the enzyme by utilizing a graphics computer. The mode of substrate binding and any conformational changes in the protein on binding the substrate will be determined from x-ray structural studies of enzyme-inhibitor complexes or a true enzyme-substrate complex stabilized at low temperatures. This structural information, when taken together with chemical and kinetic data, should indicate the kinetic mechanism of PPase.